| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
Purity: ≥98%
LY3295668 (LY-3295668; AK-01; Erbumine; AK01) is a novel, potent, orally bioavailable and highly selective Aurora-A kinase inhibitor with potential anticancer activity. It inhibits AurA and AurB with Ki values of 0.8 nM and 1038 nM, respectively. LY3295668 can kill Rb-deficient cancer cells at doses that have minimal effects on normal cells. In a kinome-wide survey, only 5 of 386 kinases are potently inhibited by LY3295668
| Targets |
Aurora A Kinase (AURKA). LY3295668 inhibits AURKA autophosphorylation in NCI-H446 cells with an IC50 of 0.46 nmol/L. It exhibits over 1,000-fold selectivity against AURKB (measured by inhibition of phospho-histone H3). [1]
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| ln Vitro |
LY3295668 is an extremely strong mutation affecting Aurora-A, having Ki values of 0.8 nM for AurA and 1038 nM for AurB. Strongly powerful AurA mutation LY3295668 has negligible effects on normal cells. Only 5 out of 386 variations were successfully suppressed by LY3295668 (<10 nM) in a panel survey [1].
Antiproliferative Activity & RB1 Synthetic Lethality: In a 2DT cell proliferation assay, LY3295668 demonstrated potent cytotoxic activity against RB1-mutant cancer cells (e.g., small-cell lung cancer, triple-negative breast cancer), while predominantly exhibiting cytostatic effects on RB1 wild-type cells. Across a panel of 517 cancer cell lines, RB1 mutation was the most significantly associated genetic event with sensitivity to LY3295668. [1] Induction of Apoptosis: LY3295668 significantly activated Caspase-3/7 and TUNEL-positive signals in RB1-null cells (NCI-H446 SCLC, MDA-MB-468 TNBC), indicating induction of apoptosis. This effect was weaker in RB1-positive cells (e.g., DMS-53, MDA-MB-231). [1] Effects on Normal Cells: Compared to RB1-mutant cancer cells, human bone marrow mononuclear cells were approximately 10-fold less sensitive to the cytotoxic effects of LY3295668 (after a 48-hour treatment, the IC50 ratio for BM cells was 10-fold higher than for NCI-H446 cells). [1] Mechanism of Action: In RB1-null cells, LY3295668 treatment led to increased stability of cyclin B1, dependent on mitotic checkpoint complex (MCC) proteins BUBR1 and MAD2, and caused a profound mitotic arrest. Knockdown of BUB1B or MAD2L1 by siRNA reversed these effects and reduced apoptosis, indicating that an intact spindle assembly checkpoint is required for LY3295668 cytotoxicity. [1] Resistance Studies: In MDA-MB-361 breast cancer cells that developed acquired resistance to the CDK4/6 inhibitor palbociclib through long-term drug selection, RB1 expression was reduced, and sensitivity to LY3295668 was markedly enhanced. In another palbociclib-resistant MCF7 cell line where RB1 levels remained unchanged, no increased sensitivity to LY3295668 was observed. [1] |
| ln Vivo |
In Vivo Antitumor Efficacy: In nude mice bearing RB1-null small-cell lung cancer xenografts (NCI-H69, LXFS 615, LXFS 650), continuous oral administration of LY3295668 (50 mg/kg, twice daily) led to significant tumor regression. In contrast, no tumor regression was observed in the RB1 wild-type SCLC PDX model LXFS-1129 at the same dose. [1]
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| Enzyme Assay |
Kinase Selectivity Profiling: The selectivity of LY3295668 was assessed by surveying 386 kinases in a kinome-wide panel. The results showed that only 5 kinases were potently inhibited by LY3295668 (IC50 < 10 nmol/L). In enzyme assays, AURKC was more than two orders of magnitude less sensitive to LY3295668 than AURKA. [1]
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| Cell Assay |
2DT Cell Proliferation Screening: To mitigate growth rate bias, the treatment duration for each cell line was set based on its average population doubling time (DT), ensuring cells underwent approximately 2 doublings during treatment. Cells in 384-well plates were treated with LY3295668 (10-point, 1:3 serial dilution starting from 10 μM) for 2DT, and cell viability was assessed using CellTiter-Glo (CTG) reagent to calculate absolute IC50 values. [1]
Apoptosis Detection by High Content Imaging: Following treatment with LY3295668, cells were fixed with formaldehyde, permeabilized with Triton X-100, and blocked with BSA. Cells were then incubated with primary antibodies against cleaved caspase-3 or TUNEL, followed by Alexa-647-conjugated secondary antibody and Hoechst 33342 or DAPI for nuclear staining. Images were acquired using a high-content imager (e.g., CellInsight NXT or Acumen eX3) to quantify the percentage of positive cells. [1] Real-Time Apoptosis Monitoring (IncuCyte): NCI-H446 and Calu-6 cells were seeded in 96-well plates and treated with various concentrations of LY3295668 for 24, 48, and 72 hours. A Caspase-3/7 reagent was added, and green fluorescent images were acquired every 2 hours using an IncuCyte Zoom live-cell imaging system to monitor caspase-3/7 activation in real-time. [1] Gene Knockdown Experiments: Cells were transfected with shRNA or siRNA targeting RB1, BUB1B, or MAD2L1. Knockdown efficiency was verified by Western blot. Following knockdown, cells were treated with LY3295668, and cell viability was assessed by cell counting, PI staining, or CTG assay, while protein expression changes (e.g., cyclin B1, cleaved PARP) were analyzed by Western blot. [1] Western Blot Analysis: Cells were lysed in lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined, and proteins were separated by SDS-PAGE and transferred to membranes. Immunoblotting was performed using specific antibodies against RB1, AURKA, p-AURKA (Thr288), cleaved caspase-3, cyclin B1, MAD2, BUBR1, etc., to detect protein expression levels and phosphorylation status. [1] |
| Animal Protocol |
In Vivo Efficacy Study: NCI-H69 SCLC cells were implanted subcutaneously into the rear flank of athymic nude female mice (7-8 weeks old). When tumors reached 80-200 mm³, mice were randomized (n=10 for treatment groups, n=8 for vehicle). LY3295668 was administered orally at 50 mg/kg twice daily (b.i.d.) continuously for at least 3 weeks. Tumor volumes were measured weekly (volume = length × width² × 0.536). [1]
Patient-Derived Xenograft Study: SCLC PDX models (LXFS 615, LXFS 650, LXFS 1129), derived from patient surgical specimens, were implanted subcutaneously into nude mice. When tumors reached 80-200 mm³, mice were randomized. LY3295668 was administered orally at 50 mg/kg twice daily (b.i.d.). The standard-of-care group received etoposide (30 mg/kg, s.c., Q7Dx4) combined with cisplatin (3.2 mg/kg, s.c., Q7Dx4). Tumor volumes were measured weekly. [1] |
| ADME/Pharmacokinetics |
Drug Exposure: In mice dosed orally with LY3295668 at 50 mg/kg twice daily, the mean free blood concentration exceeded the IC90 for AURKA inhibition throughout the dosing interval, while even at peak concentration (Cmax), it did not reach the IC90 for AURKB inhibition. In a 1-month rat toxicology study, continuous dosing achieved steady-state plasma concentrations comparable to those in the mouse xenograft efficacy studies. [1]
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| Toxicity/Toxicokinetics |
Myelosuppression: In a 1-month rat toxicology study, continuous dosing of LY3295668 (achieving steady-state plasma concentrations comparable to those effective in mouse xenograft models) caused no histologic changes in the bone marrow of the femur and sternum. Minimal changes were observed in absolute numbers of circulating red blood cells, total white blood cells, lymphocytes, and eosinophils, with no effect on reticulocytes, platelets, neutrophils, or monocytes. [1]
Preclinical Tolerability: In mice, the dosing regimen of LY3295668 at 50 mg/kg twice daily was well tolerated. [1] |
| References | |
| Additional Infomation |
LY3295668, an Aurora A kinase inhibitor, is an orally bioavailable serine/threonine protein kinase inhibitor with potential antimitotic and antitumor activities. After administration, LY3295668 targets and inhibits the activity of Aurora A kinase. This may lead to mitotic spindle assembly disorder, chromosome segregation disorder, cell division inhibition, and induction of apoptosis in cells overexpressing Aurora A kinase. Aurora A kinase is overexpressed in various cancers and plays a crucial role in the regulation of spindle assembly and mitosis.
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| Molecular Formula |
C24H26CLF2N5O2
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| Molecular Weight |
489.9454
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| Exact Mass |
489.174
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| Elemental Analysis |
C, 58.84; H, 5.35; Cl, 7.24; F, 7.76; N, 14.29; O, 6.53
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| CAS # |
1919888-06-4
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| Related CAS # |
1919888-07-5 (LY3295668 erbumine)
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| PubChem CID |
121333423
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
2.4
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
34
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| Complexity |
708
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| Defined Atom Stereocenter Count |
2
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| SMILES |
ClC1=CC=CC(=C1F)CN1CC[C@](C(=O)O)(CC2=C(C=CC(NC3C=C(C)NN=3)=N2)F)C[C@H]1C
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| InChi Key |
YQQZZYYQTCPEAS-OYLFLEFRSA-N
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| InChi Code |
InChI=1S/C24H26ClF2N5O2/c1-14-10-21(31-30-14)29-20-7-6-18(26)19(28-20)12-24(23(33)34)8-9-32(15(2)11-24)13-16-4-3-5-17(25)22(16)27/h3-7,10,15H,8-9,11-13H2,1-2H3,(H,33,34)(H2,28,29,30,31)/t15-,24-/m1/s1
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| Chemical Name |
(2R,4R)-1-(3-chloro-2-fluorobenzyl)-4-((3-fluoro-6-((5-methyl-1H-pyrazol-3-yl)amino)pyridin-2-yl)methyl)-2-methylpiperidine-4-carboxylic acid
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| Synonyms |
LY3295668; AK-01; (2R,4R)-1-[(3-chloro-2-fluorophenyl)methyl]-4-({3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]pyridin-2-yl}methyl)-2-methylpiperidine-4-carboxylic acid; (2R,4R)-1-[(3-chloro-2-fluorophenyl)methyl]-4-[[3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]pyridin-2-yl]methyl]-2-methylpiperidine-4-carboxylic acid; (2R,4R)-1-((3-chloro-2-fluorophenyl)methyl)-4-((3-fluoro-6-((5-methyl-1H-pyrazol-3-yl)amino)pyridin-2-yl)methyl)-2-methylpiperidine-4-carboxylic acid; RefChem:110363; ...; LY-3295,668; LY-3295668; AK01; LY 3295668; AK 01;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~204.10 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.5 mg/mL (5.10 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: ≥ 0.5 mg/mL (1.02 mM) (saturation unknown) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one),clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0410 mL | 10.2051 mL | 20.4102 mL | |
| 5 mM | 0.4082 mL | 2.0410 mL | 4.0820 mL | |
| 10 mM | 0.2041 mL | 1.0205 mL | 2.0410 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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